Compositions and methods for the in situ delivery of therapeutic and diagnostic agents

Abstract

Synthetic minimal cells (SMC) are provided for delivering a therapeutic or diagnostic agent to a subject or to a site within a subject in need of treatment or diagnosis. SMCs may be targeted to the site or sense the site and initiate production and release of the therapeutic or diagnostic agent. SMCs comprise a sensor, at least one genetic circuit and outputting means to deliver the therapeutic agent.

What is claimed is: 
     
         1 . A synthetic minimal cell (SMC) or a consortium of SMCs for the production and delivery of a therapeutic or diagnostic agent, wherein each SMC comprises:
 (a) at least one sensor that detects at least one condition under which the therapeutic or diagnostic agent is to be produced and delivered;   (b) at least one genetic circuit that controls the production of the therapeutic or diagnostic agent upon detection of the at least one condition by the sensor; and   (c) at least one outputting means for delivering the therapeutic or diagnostic agent outside of the SMC.   
     
     
         2 . A synthetic minimal cell (SMC) or a consortium of SMCs for the delivery of a therapeutic or diagnostic agent, wherein each SMC comprises:
 (a) a therapeutic or diagnostic agent;   (b) at least one sensor that detects at least one condition under which the therapeutic or diagnostic agent is to be delivered; and   (b) at least one genetic circuit that controls the production of an outputting agent for delivering the therapeutic or diagnostic agent outside the SMC upon detection of the at least one condition by the sensor.   
     
     
         3 . The SMC of  claim 1  or  2  wherein the therapeutic, diagnostic or outputting agent is a protein, peptide, nucleic acid or small molecule. 
     
     
         4 . The SMC of  claim 3  wherein the nucleic acid is DNA, RNA, shRNA, siRNA, CRISPR sgRNA or an antisense oligonucleotide. 
     
     
         5 . The SMC of  claim 3  wherein the agent is a protein or nucleic acid enzyme, antibody, antibody fragment, catalytic peptide, antibiotic, antimicrobial peptide, membrane-disrupting protein, transporter, signal protein, cytokine or chemokine. 
     
     
         6 . The SMC of  claim 1  or  2  wherein the therapeutic or diagnostic agent is not produced or delivered until the genetic circuit activates production of the therapeutic agent, diagnostic or outputting agent or a precursor thereof within the SMC. 
     
     
         7 . The SMC of  claim 1  or  2  wherein the sensor is membrane-bound, is soluble within the SMC, or comprises a membrane sensitive to the detected condition. 
     
     
         8 . The SMC of  claim 1  or  2  wherein the sensor detects one or more conditions that exist at a site where delivery of the therapeutic or diagnostic agent is desired. 
     
     
         9 . The SMC of  claim 8  wherein the conditions exist at the site is a result of the disease to which the therapeutic agent is desirously delivered. 
     
     
         10 . The SMC of  claim 8  wherein the one or more conditions are created at the site where the therapeutic agent is beneficially delivered. 
     
     
         11 . The SMC of  claim 10  wherein the condition is selected from radiation, heat, pH change, or the administration of an agent that targets the site desirous of beneficial therapeutic agent release and activates the SMC sensor, production, outputting or any combination thereof. 
     
     
         12 . The SMC of  claim 1  or  2  wherein the production comprises translation. 
     
     
         13 . The SMC of  claim 1  or  2  wherein the production comprises gene expression. 
     
     
         14 . The SMC of  claim 1  or  2  wherein the production is controlled by transcription factors. 
     
     
         15 . The SMC of  claim 1  wherein the outputting means is by passive or controlled release from the SMC. 
     
     
         16 . The SMC of  claim 1  wherein the outputting means is responsive to a condition different from that of the sensor. 
     
     
         17 . The SMC of  claim 1  or  2  further comprising a negative feedback sensor that reduces production or outputting of the therapeutic agent. 
     
     
         18 . The SMC of  claim 1  or  2  wherein the sensor means, production means and outputting means of the therapeutic or diagnostic agent occurs in one, or two or three different SMCs. 
     
     
         19 . The SMC of  claim 2  wherein the outputting agent is a protein, a peptide, a small molecule, a membrane channel polypeptide (also referred to herein as a “pore”), a membrane pump polypeptide, a trafficking polypeptide, a signal polypeptide or an export polypeptide. 
     
     
         20 . The SMC of  claim 1  or  2  comprising at least a portion of at least one multi-gene genetic circuit. 
     
     
         21 . The SMC of  claim 1  or  2 , wherein the SMC comprises at least one multi-gene genetic circuit. 
     
     
         22 . The SMC of  claim 1  or  2 , wherein the SMC comprises a portion of a multi-gene genetic circuit and a second SMC includes a second portion of the multi-gene genetic circuit. 
     
     
         23 . The SMC of  claim 1  or  2 , wherein the multi-gene genetic circuit comprises 2, 3, 4, or more gene components. 
     
     
         24 . The SMC of  claim 1  or  2 , wherein the portion of the multi-gene genetic circuit comprises 1, 2, 3, 4 or more gene components. 
     
     
         25 . The SMC of  claim 23  or  24 , wherein contacting the SMC with an activator modulates an activity of at least one gene component of the genetic circuit. 
     
     
         26 . The SMC of any one of  claim 23  or  24 , wherein an activity of a first gene component of the SMC modulates an activity of one or more additional gene components of at least one of: (1) the multi-gene genetic circuit of the SMC and (2) a multi-gene genetic circuit of another SMC. 
     
     
         27 . The SMC of  claim 26 , wherein the multi-gene genetic circuit of (2) is different than the multi-gene genetic circuit of (1). 
     
     
         28 . The SMC of any one of  claims 23 - 27 , wherein an activity of the multi-gene circuit comprises expression of 1, 2, 3, 4, or more polypeptides encoded by the gene components. 
     
     
         29 . The SMC of  claim 25 , wherein the activator is a small molecule and optionally is soluble. 
     
     
         30 . The SMC of  claim 25 , wherein the activator is present on or proximal to a disease site within the body. 
     
     
         31 . The SMC of any one of  claims 20 - 30 , wherein an activity of a first multi-gene genetic circuit modulates an activity of at least one additional multi-gene genetic circuit, and optionally activates a cascade of activity of 1, 2, 3, 4, or more additional gene components of the multi-gene genetic circuit in the SMC or in at least one additional SMC. 
     
     
         32 . The SMC of any one of  claims 20 - 30 , wherein the SMC comprises one or more of prokaryotic or eukaryotic transcription/translation (TX/TL) components. 
     
     
         33 . The SMC of any one of  claims 20 - 30 , wherein the SMC comprises one or more expression vectors comprising one or more of the gene components. 
     
     
         34 . The SMC of  claim 33 , wherein the expression vector comprises one or more of: a promoter sequence and a polynucleotide sequence encoding a polypeptide. 
     
     
         35 . The SMC of  claim 34 , wherein the polynucleotide sequence encodes at least one of a membrane channel polypeptide and a detectable label polypeptide. 
     
     
         36 . The SMC of any one of  claims 20 - 30 , wherein the SMC comprises a fusion-inducing polypeptide in association with the SMC's exterior surface. 
     
     
         37 . The SMC of  claim 36 , wherein the fusion-inducing polypeptide is a SNARE polypeptide or a SNARE polypeptide mimic. 
     
     
         38 . The SMC of any one of  claims 20 - 30 , wherein the SMC is fused to at least a second SMC comprising at least one independently selected multi-gene genetic circuit. 
     
     
         39 . The SMC of  claim 38 , wherein the SMC and the second SMC comprise the independently selected multi-gene genetic circuit. 
     
     
         40 . The SMC of  claim 38 , wherein the SMC does not comprise the independently selected multi-gene genetic circuit of the second SMC. 
     
     
         41 . A composition comprising a plurality of the SMCs of any one of  claims 1 - 40 , wherein the multi-gene genetic circuits of the SCMs are independently selected. 
     
     
         42 . The composition of  claim 41 , wherein the multi-gene genetic circuit of the SMCs comprises 1, 2, 3, 4, or more independently selected gene components. 
     
     
         43 . The composition of  claim 41  or  42 , wherein the SMCs in the plurality comprise the same multi-gene genetic circuit. 
     
     
         44 . The composition of  claim 41  or  42 , wherein the SMCs in the plurality comprise independently selected multi-gene genetic circuits. 
     
     
         45 . The composition of any one of  claims 40 - 44 , wherein contacting an SMC of the plurality of SMCs with an externally delivered agent modulates an activity of at least one gene component of the multi-gene genetic circuit of the contacted SMC. 
     
     
         46 . The composition of any one of  claims 41 - 44 , wherein at least one of the SMCs in the plurality of SMCs is fused to another of the SMCs in the plurality of SMCs. 
     
     
         47 . The composition of any one of  claims 41 - 43 , wherein one or more multi-gene genetic circuits in two or more SMCs of the plurality of SMCs are active in parallel. 
     
     
         48 . The composition of any one of  claims 41 - 45 , wherein an activity of one or more multi-gene genetic circuits in a first SMC of the plurality is modulated by at least one of: (1) an activity of a multi-gene genetic circuit in the first SMC of the plurality; and (2) an activity of a multi-gene genetic circuit in a second SMC of the plurality. 
     
     
         49 . The composition of any one of  claims 41 - 48 , wherein two or more of the plurality of SMCs operate in conjunction with each other as a network. 
     
     
         50 . The composition of  claim 49 , wherein operating in conjunction with each other comprises being in chemical communication with each other. 
     
     
         51 . The composition of any one of  claims 39 - 48 , wherein an activity of the multi-gene genetic circuit comprises expression of 1, 2, 3, 4, or more polypeptides. 
     
     
         52 . The composition of any one of  claims 39 - 49 , wherein an activity of a first gene component of an SMC of the plurality of SMCs modulates an activity of one or more additional gene components of at least one of: (1) the multi-gene genetic circuit of the SMC and (2) a multi-gene genetic circuit of another SMC of the plurality of SMCs. 
     
     
         53 . The composition of  claim 50 , wherein the multi-gene genetic circuit of (2) is different than the multi-gene genetic circuit of (1). 
     
     
         54 . The composition of any one of  claims 39 - 51 , wherein an activity of a first multi-gene genetic circuit of an SMC of the plurality of SMCs modulates an activity of at least one additional multi-gene genetic circuit of an SMC of the plurality of SMCs, and optionally activates a cascade of activity of 1, 2, 3, 4, or more additional gene components of the first multi-gene genetic circuit in the SMC or in at least one additional SMC in the plurality of SMCs. 
     
     
         55 . The composition of any one of  claims 39 - 52 , wherein the plurality of SMCs comprises one or more of: bacterial transcription/translation (TX/TL) components and mammalian TX/TL components. 
     
     
         56 . The composition of any one of  claims 39 - 66 , wherein the plurality of SMCs comprises one or more independently selected expression vectors. 
     
     
         57 . The composition of  claim 54 , wherein the expression vector comprises one or more of: a promoter sequence and a polypeptide-encoding polynucleotide sequence. 
     
     
         58 . The composition of  claim 55 , wherein the polynucleotide sequence encodes at least one of: a membrane channel polypeptide and a detectable label polypeptide. 
     
     
         59 . The composition of any one of  claims 39 - 56 , wherein at least a portion of the SMCs in the plurality of SMCs comprise a fusion-inducing polypeptide in association with the SMCs' exterior surfaces. 
     
     
         60 . The composition of  claim 57 , wherein the fusion-inducing polypeptide is a SNARE polypeptide or a SNARE polypeptide mimic. 
     
     
         61 . The composition of  claim 58 , wherein the SNARE polypeptide or SNARE polypeptide mimic associated with the exterior surface of the SMCs in a first portion of the plurality of SMCs that comprise a fusion-inducing polypeptide, is complementary to the SNARE polypeptide or SNARE polypeptide mimic associated with the exterior surface of the SMCs in a second portion of the plurality of SMCs. 
     
     
         62 . The composition of any one of  claims 39 - 60 , wherein an activity of a first multi-gene genetic circuit in one or more SMCs of the plurality of SMCs activates at least one additional multi-gene genetic circuit in one or more SMCs of the plurality of SMCs. 
     
     
         63 . The composition of  claim 60 , wherein an activity of a multi-gene genetic circuit in an SMC of the plurality of SMCs results in a cascade of multi-gene genetic circuit activation in one or more SMCs of the plurality of SMCs. 
     
     
         64 . The composition of any one of  claims 39 - 61 , wherein an activity of a first multi-gene genetic circuit in a first SMC of the plurality of SMCs activates 1, 2, 3, 4, or more additional multi-gene genetic circuits in one or more of: (1) the first SMC and (2) a second SMC of the plurality of SMCs. 
     
     
         65 . The composition of  claim 62 , wherein the additional multi-gene genetic circuit is selected from: (1) a genetic circuit that is the same as the first multi-gene genetic circuit and (2) a multi-gene genetic circuit that is different than the first multi-gene genetic circuit. 
     
     
         66 . The composition of any one of  claims 39 - 63 , wherein contacting at least one SMC of the plurality of SMCs with an externally delivered agent modulates an activity of at least one of the multi-gene genetic circuits of the contacted SMC. 
     
     
         67 . The composition of any one of  claims 39 - 64 , wherein an activity of a multi-gene genetic circuit of an SMC of the plurality of SMCs results in contacting one or more multi-gene genetic circuits of the SMC with an agent that modulates an activity of the one or more multi-gene genetic circuits. 
     
     
         68 . A method for treating a condition or disease that benefits from a therapeutic agent comprising administering to a subject in need thereof a SMC of  claim 1  or  claim 2 . 
     
     
         69 . The method of  claim 68  wherein the SMC targets a site of disease. 
     
     
         70 . The method of  claim 68  wherein the disease is cancer, thrombosis or enzyme deficiency. 
     
     
         71 . The method of  claim 68  wherein the treating comprises:
 (a) administering a SMC comprising a sensor that detects an externally applied signal; and 
 (b) applying the signal to the part of the body in which delivery of the therapeutic agent is desired. 
 
     
     
         72 . The method of  claim 68  wherein the therapeutic agent is a protein, peptide, nucleic acid or small molecule. 
     
     
         73 . The method of  claim 68  wherein the nucleic acid is DNA, RNA, shRNA, siRNA, an antisense oligonucleotide, a microRNA inhibitor, an anti-miRNA or a sgRNA. 
     
     
         74 . The method of  claim 68  wherein the protein is an antibody, toxin, hormone, marker or an enzyme. 
     
     
         75 . The method of  claim 68  wherein the peptide is an antibiotic or antimicrobial peptide. 
     
     
         76 . A method for diagnosing a condition or disease comprising administering to a subject in need thereof a SMC of  claim 1  or  claim 2 . 
     
     
         77 . The method of  claim 76  wherein the SMC targets a site of disease. 
     
     
         78 . The method of  claim 76  wherein the disease is cancer, thrombosis or enzyme deficiency. 
     
     
         79 . The method of  claim 6  wherein the diagnosing comprises:
 (a) administering a SMC comprising a sensor that detects an externally applied signal; and 
 (b) applying the signal to the part of the body in which diagnosis of the disease is desired. 
 
     
     
         80 . The method of  claim 76  wherein the diagnostic agent is a protein, peptide, nucleic acid or small molecule. 
     
     
         81 . The method of  claim 76  wherein the nucleic acid is DNA, RNA, shRNA, siRNA, an antisense oligonucleotide, a microRNA inhibitor, an anti-miRNA or a sgRNA. 
     
     
         82 . The method of  claim 76  wherein the protein is an antibody, toxin, hormone, marker or an enzyme. 
     
     
         83 . The method of  claim 76  wherein the peptide is an antibiotic or antimicrobial peptide. 
     
     
         84 . The method of  claim 76  wherein the diagnostic agent is detected in a bodily fluid of the subject. 
     
     
         85 . The method of  claim 84  wherein the bodily fluid is blood, urine, saliva, cerebrospinal fluid, ascites, or lymphatic fluid. 
     
     
         86 . The method of  claim 76  wherein the diagnostic agent is detected by imaging.